IS 643 

B4 
Copy 1 



A CORRELATION BETWEEN BACTERIAL ACTIVITY 
AND LIME REQUIREMENT OF SOILS 



FIRMAN E. BEAR 



±au 



Department of Agricultural Chemistry and Soils, Ohio State University 



Reprinted from 

Soil Science, Vol. IV, No. 6, December, 1917 






Reprinted from Soil Science, 
Vol. IV, No. 6, December, 1017 



A CORRELATION BETWEEN BACTERIAL ACTIVITY AND LIME 
REQUIREMENT OF SOILS 

FIRMAN E. BEAR 

Department of Agricultural Chemistry and Soils, Ohio State University 

Received for publication September 12, 191? 

INTRODUCTION 

Limestone regions are noted for their fertility. Alfalfa, red clover, blue- 
grass, and corn are among the crops which thrive best on limestone soils. 
Those soils which do not naturally contain carbonate of lime are usually made 
more productive by applications of lime or limestone. Extensive investiga- 
tions carried out by the Rhode Island, Maryland, Pennsylvania, Ohio, Illi- 
nois, and other agricultural experiment stations have demonstrated the 
value of lime in either the oxide, hydrate or carbonate form on soils which 
are acid to litmus. An excellent review of the most important investigational 
work on the use of lime on acid soils is given by Frear (9). 

The investigations of Wheeler at the Rhode Island Agricultural Experi- 
ment Station, indicate, however, that a number of plants of economic im- 
portance thrive on soils which contain no solid carbonate of lime. Some of 
these plants are benefited by lime, but others are injured by applications of 
lime. Wheeler (36) says that orchard grass (Dactylis glomerate, L.) and mea- 
dow fescue (Festuca elatior, L.) are less injured by soil acidity than Kentucky 
blue-grass (Poa pratensis, L.) and timothy (Phleum pratense, L.) and that 
awnless brome grass (Bromus inermus, L), red top {Agrostis alba var. vulgaris, 
Thurb.), and Rhode Island bent (Agrostis canina, L.) do not seem to be suscep- 
tible to injury even on decidedly acid soils. He also states (37) that Concord 
grapes are apparently indifferent to the lack of lime and that cranberries, 
raspberries, and lima beans are injured by liming, the last named growing 
splendidly on soils so acid as to entirely destroy lettuce, spinach, onions, beets 
and asparagus. In ln> latest publication on this subject Wheeler (38) gives 
a summary of his work in which he shows that plants vary in their require- 
ments from those which are injured by applications of lime even to a very 
acid soil, to those which are unable to live on an acid soil and are benefited 
remarkably by lime. 

Coville (6) states that the blueberry, cranberry, strawberry, blackberry, 
red respberry, potato, sweet potato, rye, oats, millet, buckwheat, red top, 
carrot, turnip, cowpea, hairy vetch, crimson clover, soybean, lupine, and 
serradella are adapted to acid soils. He concludes, "soil acidity is not always 

433 

SOIL SCIENCE, VOL. IV, NO. 6 



434 FIRMAN E. BEAR 

an objectionable condition which invariably requires lime" and "under cer- 
tain conditions, a complete system of acid agriculture is practicable." 

Harter (14) writes that liming has been shown to be beneficial to all crops 
on Norfolk sqils with the exception of beans, peas, and tomatoes. Kossovitch 
and Althausen (26) report that, while the liming of acid podzol soils strikingly 
increases the yields, the limit of increase is at about the point of neutralization 
and that an excess injures the plants. No statement is made as to how the 
point of neutralization was determined. Heinrich (15) concludes that the 
determination of lime in a soil, by digesting with 10 per cent hydrochloric 
acid, can be used as an index in determining what crops will thrive. Ac- 
cording to his report, the least amounts of lime which will permit of successful 
growth are : 

Calcium carbonate 
Crops * n the soil 

per cent 

Lupines, potatoes, and rye . 05 

Oats and barley 0.05 to 0. 10 

Peas and vetch 0.10 

Red clover 0. 10 to 0. 12 

Alfalfa 0.20 to 0.30 

Fred and Graul (10) experimenting with alfalfa, soybeans, and red clover 
on acid soils of two series, conclude that half enough lime to neutralize the 
soil acidity as measured by the Truog (32) method is sufficient for the pro- 
duction of good yields of these crops on acid soils of these two series. 

THE RELATION BETWEEN BACTERIAL ACTIVITY AND THE REACTION OF SOILS 

One of the reasons usually given for the maintenance of a neutral or slightly 
alkaline reaction in soils is that the soil microorganisms, which have to do with 
the processes of decay and the changes by which certain organic and inorganic 
substances become available for higher plants are unable to work to best 
advantage in an acid medium. The ammonifying, nitrifying, and nitrogen- 
fixing bacteria are thought to prefer a neutral or slightly alkaline medium. 
However, it is probably true that the various groups of soil bacteria are differ- 
ently affected by the soil reaction. The influence of acidity and alkalinity 
on the development of pathogenic bacteria has been studied by a number of 
investigators. The literature on this subject is reviewed quite fully by Itano 
(21). The degree of acidity or alkalinity which the organisms are able to 
withstand varies with the species. Certain forms, e.g., Bacterium tuberculosis, 
are able to live in the presence of a considerable degree of acidity. It is 
reasonable to believe that soil microorganisms show similar differences in this 
respect. The fact that many acid soils are supporting vegetation, indicates 
that bacterial processes are being carried on in them, although these processes 
might be materially hastened if lime were applied. 

The number of bacterial colonies from soil aliquots which will develop on 



41 5 iff» 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 435 

agar plates is influenced by the reaction of the medium. Hoffmann (16) 
finds in counting the number of bacteria in soils that a medium slightly acid 
to phenolphthalein is more favorable than a medium which is neutral or 
slightly alkaline to phenolphthalein. Fischer (8), who conducted probably 
the most extensive investigations on the effect of lime on the number of bac- 
teria in soils, shows that an application of either calcium oxide or calcium 
carbonate has a very marked effect in increasing the total number of bacteria. 

That the rate of ammonification is increased by applications of lime is 
shown by Voorhees and Lipman (35). Coville (6) points out that many soils 
acid to litmus contain large amounts of ammonia. Kopeloff (25) shows' that 
"where the soil reaction is unfavorable for the activities of the soil bacteria 
concerned in ammonification, the soil fungi may prove to be an important 
compensating factor." 

The rate of nitrification is increased by applications of lime on soils which 
give an acid reaction with litmus. The results obtained by Lyon and Bizzell 
(27) are typical. A number of other investigators report similar effects from 
the use of lime. Scales (29), studying the activities of nitrifying organisms, 
finds they are most active in the presence of 50 per cent of the calcium-car- 
bonate requirement (Veitch) of the soil. An excess of calcium carbonate 
seems to be toxic to the nitrifying organisms. Temple (31) finds that if an 
organic source of nitrogen is used instead of ammonium sulfate, the formation 
of nitrates is much greater in acid soils. He explains this increased nitri- 
fication on the basis of the formation of neutral zones, caused by the production 
of ammonia, at which points conditions are favorable for nitrification. Temple 
also shows that calcium salts of organic acids can be used as effectively as 
calcium carbonate in overcoming the toxic effect of ammonium sulfate on an 
acid soil. Miller (28), working with a sandy soil acid to litmus, finds that an 
application of 0.1 per cent of calcium oxide caused a decrease in the ability 
of the soil to nitrify ammonium sulfate and that 0.5 per cent of calcium oxide 
stopped the process entirely. Hutchinson (19) finds that calcium oxide acts 
not alone as a neutralizing agent, but also as a partial sterilizing agent. Since 
in the experimental work following applications of neutralizing agents are 
confined to calcium carbonate, it does not seem necessary to include any 
further discussion on the effect of calcium oxide on the bacterial processes in 
the soil. 

It should be remembered that it has been shown that nitrate nitrogen is 
not necessary for all plants. Hall and Miller (12) call attention to the fact 
that ammonium sulfate, on the Park plats of the Rothamsted Farm, pro- 
duces very good crops of grass, although the soil is deficient in lime and very 
little nitrification takes place. Hutchinson and Miller (20) find that peas 
are able to utilize ammonia nitrogen as well as nitrate nitrogen, although the 
opposite is true with wheat. Kelley (24) shows that rice, grown in swamp 
land, secures its nitrogen in the form of ammonia. If ammonification proc- 
esses are less affected than nitrification processes by a deficiency of lime in 



436 FIRMAN E. BEAR 

the soil, then plants which are able to utilize ammonia can survive where those 
depending on nitrate nitrogen cannot live. 

Hopkins (18) notes that the application of lime increases the power of 
Bacillus radicicola in certain legumes to fix atmospheric nitrogen. Whiting 
(39) writes that nodules are often found in abundance on legumes on very 
acid soils. Japanese clover (Lespedeza) has often been observed by the writer 
growing on soils strongly acid to litmus and the roots were well supplied with 
nodules. These nodules were mostly near the surface of the soil. Keller- 
man and Robinson (22) find that crimson clover inoculation is little affected 
by the reaction of the soil. Fred and Graul (10) find that, if acid Colby silt 
loam soil is previously inoculated with B. radicicola, nitrogen fixation by soy- 
beans is little influenced by applications of calcium carbonate. They also 
find this true on acid Colby silt loam with red clover. Both clover and alfalfa 
were able to fix considerable amounts of nitrogen when growing on Colby 
silt loam and Plainfield sand having only one-half of their acidity (Truog 
method) neutralized. The Colby silt loam required 10,400 and the Plain- 
field sand 5200 pounds of calcium carbonate to neutralize one-half of the 
acidity in 2,000,000 pounds of soil. Determinations of the lime requirement 
(Veitch) on the Colby silt loam soil, chosen from the same locality the year 
previous, showed a need of 3234 pounds of calcium carbonate per 2,000,000 
pounds of soil. The authors state that "the Truog method shows much larger 
amounts of soil acidity than the Veitch." 

Ashby (1) shows that the use of lime on the Rothamsted soils more than 
doubled the nitrogen-fixing power of the Azotobacter. Hoffman and Hammer 
(17) find that calcium carbonate is essential to non-symbiotic nitrogen fixa- 
tion, but that the amount required is very minute and was present in 
sufficient amount in all the soils tested. These soils were chosen from various 
localities in Wisconsin and must have included some soils acid to litmus, since 
Whitson and Weir (40) estimate that two-thirds of the soils of Wisconsin 
are acid. Christensen and Larsen (4) find that if Ashby 's solution is inocula- 
lated with a soil in need of lime, the brownish film usually produced by Azoto- 
bacter does not develop. They suggest this as a method of determining the 
need of a soil for lime. 

Gimingham (11) describes several organisms capable of bringing about the 
formation of carbonates from calcium salts of organic acids. Hall and Miller 
(13) also report that calcium salts of organic acids are transformed to the 
carbonate by soil organisms, the organic acids being decomposed to form carbon 
dioxide and water. Drew (7) shows that marine bacteria precipitate calcium 
carbonate from sea water. He names the organism responsible for this re- 
action, Bacillus calcis. Kellerman and Smith (23) write that it is possible 
in the laboratory to produce calcium carbonate by three types of biological 
processes; by the action of ammonium carbonate on calcium sulfate; by the 
action of ammonium hydroxide on calcium acid carbonate, and by the de- 
composition of calcium salts of organic acids. They state that Drew's organ- 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 437 

ism is Pseudomonas calcis. This is a denitrifying organism which produces 
ammonia by the reduction of nitrates. Bear and Salter (2) show that the 
lime requirement (Veitch) of the West Virginia Agricultural Experiment 
Station fertility plots is less where the content of organic matter has been 
increased, and suggest that this decrease may have been due to the precipita- 
tion of calcium from solution by the humus in the soil, whereby it was pre- 
vented from being lost in the drainage water. This calcium might later be 
freed as the carbonate, as the decomposition of the organic matter was brought 
about by the soil organisms. 

OBJECT OF THESE INVESTIGATIONS 

In view of the fact that large areas of land are acid and that the distance 
from the supply of lime often makes the cost of applying large amounts of 
lime or limestone prohibitive, it was thought it might be desirable to consider 
more carefully the possibilities of a system of acid agriculture as suggested 
by Coville (6). Since the problem of the economy of nitrogen and its availa- 
ability for the use of crops is largely a bacterial problem, it seemed important 
to study the relation of the reaction of the soil to the activities of the bacteria 
concerned in nitrogen accumulation and transformations. Recognizing the 
fact that plants do grow on soils which are acid to litmus, how are these plants 
supplied with nitrogen? We know that lime and limestone are valuable soil 
amendments, but might it not be possible that small applications of these 
materials would be relatively more effective in promoting the activities of the 
bacteria concerned in the nitrogen problem than large applications? If the 
B. radicicola of some legumes is more resistant to acidity than the B. radicicola 
growing on other legumes, might it not be possible to select legumes adapted 
to the reaction of the soil instead of adding lime to the soil to make the reaction 
suitable for the legumes we desire to grow? Even if nitrogen-fixing organisms 
are able to grow in acid soils, are they able to fix atmospheric nitrogen in such 
an environment? To answer these questions, it was proposed to measure 
the activities of those bacteria concerned in the nitrogen economy of plants 
as influenced by various amounts of calcium carbonate applied to acid soils. 

DEFINITION OF "LIME REQUIREMENT" 

In the preceding discussion, a rather loose construction is given to the term 
"soil acidity." This is simply in accordance with precedents set by the 
various investigators whose work is reviewed. As a rule, an "acid" soil 
means a soil which changes blue litmus paper red. The "degree of acidity" 
of soils has no such definite meaning, consequently the investigations reported 
are not strictly comparable. The writer sees no reason to disagree with Truog 
(33) as to what "soil acidity" really is. Truog writes that acid silicates are 
the main cause of soil acidity in upland soils. His excellent review of this 
subject gives a select bibliography of the investigational work along this 



438 FIRMAN E. BEAR 

line. Truog (32) also writes that the acidity of soils may be conveniently 
divided into two classes, "active" and "latent" acidity. He states that 
"latent" acidity is undoubtedly much less injurious to plants than "active" 
acidity. He also shows the desirability of knowing the "avidity" of the 
active soil acids. Sharp and Hoagland (30) attempt to measure the lime 
requirement of soils by determining the hydrogen-ion concentration of the 
soil suspensions. The recent review of Clark and Lubs (5) of the literature 
on this subject, indicates that the hydrogen-ion concentration of the medium 
is the important factor to consider in the relationship between acidity and 
biological processes. The hydrogen-ion concentration of a soil in suspension 
in water is, however, not a measure of the amount of lime necessary to add to 
an acid soil to produce a neutral reaction of the soil. This is partly because 
of the slow solubility of the acid-forming constituents present in soils. 

At the time this investigation was begun, most of the recent work on soil 
acidity had not been published. The writer felt at that time that the most 
satisfactory measure of the "lime requirement" of a soil was that obtained by 
the Veitch (34) method. Accordingly, this method was used in determining 
the quantitative need of the soils used for lime. It is interesting to note in 
this connection that when the two soils which were used most largely in these 
investigations had been treated with the quantity of calcium carbonate neces- 
sary to satisfy their lime requirements (Veitch) and had been mixed once each 
week for 12 weeks, they were found to be neutral to litmus paper. 

HISTORY OF THE SOILS USED IN THESE EXPERIMENTS 

A large part of the work reported has been done on samples of soil from two 
different localities belonging to different soil series. Both of these were acid 
in reaction, as will be shown later. 

Soil I was secured from plot 18 of the West Virginia Agricultural Experi- 
ment Station farm. The soil is classified by the United .States Bureau of 
Soils as Dekalb silt loam. It is a residual soil which has been formed by the 
disintegration of sandstone and greenish gray shales overlying the Pittsburg 
coal. The original timber was largely oak and chestnut with an occasional 
locust. The analysis of this soil is as follows: 

Pounds per 
Element 2,000,000 of soil 

Nitrogen 1,940 

Phosphorus 600 

Potassium 25,100 

Carbon 23,900 

Calcium 2,300 

Magnesium 4,300 

Calcium carbonate requirement (Veitch) 3,500 

Plot 18 has not received any fertilizer, lime or manure since the beginning 
of the fertilizer tests in 1900. OriTy a partial record of the produce of this 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



439 



plo;t i s available. During a part of the time since 1900 a tile drain, which 
passed near this plot, was not working, and, since the yields of the plot were 
somewhat abnormal, no permanent records of the plot were kept. Later the 
record of the produce of this plot was continued. This record shows that 
plot 18 corresponds normally in productivity to plot 21, which also received 
no fertilizer, lime or manure. The sample of soil was chosen from plot 18 
because its record was incomplete and any change due to the removal of a 
large sample of soil would not interfere with the plot experiments. Since 
1900 the following crops have been grown on these plots; rye, 1900 and 1907; 
wheat, 1901 and 1914; clover, 1902, 1909, and 1915; corn, 1903, 1905, and 1912; 
cowpeas, 1904; potatoes, 1906; timothy, 1909, 1910, and 1911, and oats, 1913. 
Table 1 gives the records of the fertilizer treatment and total produce of all 
the plots up to and including 1915. 

TABLE 1 

Total amounts of fertilizers applied and total produce per acre from 1900 to 1915 on soil I 



PLOT 


TREATMENT 


NITRATE 
OF SODA 


ACID 
PHOSPHATE 


SULFATE 
OF POTASH 


LIME 

(CaO) 


MANURE 


TOTAL 
PRODUCE 


19 
20 


N, P, K, CaO 
M, CaO 


pounds 

4200 

300 

4200 

4200 
4200 

4200 


pounds 

4200 

Ash of 40 

4200 
4200 

4200 

4200 


pounds 

1625 

tons of m 

1625 

1625 
1625 

1625 


pounds 

4500 
4500 

5500 
mure lintil 


tons 
210 

1912 
190 


pounds 

120,605 
152,400 


21 


Check 


38,600 


22 


CaO 


36,615 


23 
24 
25 
26 


AshM, N 

Check 

M 

N, P, K 


39,270 

43,075 

139,670 

117,910 


27 


Check 


42,170 


28 
29 


P, K 

N 


76,995 
52,215 


30 
31 


Check 

N, P 


39,480 
95,940 


32 


K 


41,565 


33 


Check 


36,845 


34 


P 


63,415 


35 


N 


41,195 









N, indicates nitrate of soda; P, acid phosphate; K, sulfate of potash; M, manure. 



Soil II was secured from the Ohio Agricultural Experiment Station farm at 
Wooster. This soil is classified by the Bureau of Soils as Wooster silt loam. 
It has been formed from the disintegration of sandstone and shales of the 
Mississippian period, under the influence of glacial action. The analysis of 
the soil used is as follows: 



440 FIRMAN E. BEAR 

Pounds per 
Element 2,000,000 of soil 

Nitrogen 1,775 

Phosphorus 664 

Potassium 34,000 

Carbon 22,200 

Calcium 4,470 

Magnesium 6,596 

Calcium carbonate requirement (Veitch) 3,500 

It will be observed that soil II has the same calcium-carbonate requirement 
as soil I. 

Soil II has never received any fertilizer, lime or manure since the beginning 
of the fertilizer tests in 1893. Continuous records since that time have been 
kept on soil of the same history as this soil in a 5-year rotation experiment 
at the Wooster station. The rotation has been corn, oats, wheat, clover, and 
timothy. A summary of the effect of lime and fertilizers on this soil is given 
by Williams (41) in table 2. An experiment has also been in progress on this 
same type of soil which had been kept in a fair state of fertility by a good 
rotation and an occasional application of manure previous to the beginning 
of the experiment. The rotation since practiced has been corn, oats, and 
clover. The records of this experiment are shown in table 3. It will be seen 
by a study of tables 2 and 3, that both lime and acid phosphate are very effec- 
tive in increasing the yields of the crops grown in these two rotations. While 
lime is very efficient, it seems remarkable that such large yields of these crops 
can be produced by the use of acid phosphate alone on a soil which has a 
calcium-carbonate requirement of 3500 pounds per 2,000,000 pounds of soil. 

The other samples of soil used in these experiments were Dekalb soils chosen 
from various localities in West Virginia. These soils vary greatly because 
of differences in the systems of management they have undergone. Analyses 
of these soils are shown in subsequent tables. 

PLAN OF THESE EXPERIMENTS 

Large samples of soils, acid to litmus, were secured, sent immediately to 
the laboratory, made to pass a 2-mm. sieve, and stored in large galvanized 
iron cans. From these cans soil was removed as needed. Careful analyses 
of the soils were made for the total amount of nitrogen, phosphorus, potassium, 
calcium, magnesium, and carbon. Lime-requirement determinations were 
made by the Veitch method as indicated above. Amounts of C. P. calcium 
carbonate varying from 250 pounds to 40,000 pounds per 2,000,000 pounds 
of soil were added to the soils. A study was made of the effects of these 
applications on: (a) the number of bacteria, (b) the rate of ammonification, 
(c) the rate of nitrification, (d) the fixation of nitrogen by non-symbiotic 
organisms, and (e) the development of B. radicicola of the soybean. All 
analyses were made according to the methods given by Bear and Salter (3). 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



441 



The calcium carbonate was applied and mixed thoroughly with the soil, 
which was then placed in 1-galion stone jars. Enough water was added to 
the soil to give it an optimum moisture content. Each week the soil was 
removed from the jars and mixed thoroughly and the loss of moisture, due to 

TABLE 2 

The effect of lime on the yields of crops on soil II 



2 

8 

11 

17 

18 
24 

26 

29 



TREATMENT 



Phosphorus* 

Phosphorus,* potassium 

Phosphorus*, potassium, ni- 
trogen 

All three with less nitrogen 
but more phosphorus*. . . . 

Barnyard manure 

Same as 17 but nitrogen in 
sulfate of ammonia 

Same as 17 but phosphorus 
in bone meal 

Same as 1 7 but phosphorus 
in basic slag 



Average unfertilized. 



YIELD PER ACRE 



Corn 
1900-1915 



bus. 

35.51 
43.95 

47.67 

47.2'3 
56.31 

46.23 

46.01 

46.27 



26.48 



bus. 

42.32 
51.08 

55.12 

55.67 
61.68 

55.98 

51.17 

51.69 



32.32 



Oats 
1901-1916 



bus. 
39.16 
42.62 

49.77 

51.84 
43.62 

48.21 

46.37 

47.77 



27.19 



bus. 

42.85 
46.38 

49.71 

52.38 
44.93 

51.36 

46.81 

47.85 



32.08 



Wheat 
1906-1916 



bus. 
21.48 
22.17 

31.27 

27.32 
29.51 

24.70 

27.78 
29.76 



12.74 



bus. 
25.17 
26.38 

31 .86 

30,85 
32.49 

31.26 

28.65 

28.93 



16.09 



Clover 
1903-1916 



lbs. 

1848 
2144 

2683 

2492 

3448 

2139 
2945 
2981 



1276 



lbs. 

2680 
3166 

3388 

3598 
4393 

3544 

3772 

3371 



1841 



Timothy 
1909-1916 



lbs. 

3058 
3125 

3445 

3364 

4525 

3111 
3504 

3741 



2500 



E 

lbs. 
3810 
3881 

4124 

4543 
5531 

4409 

4585 

4306 

3069 



Phosphorus in the form of acid phosphate. 



TABLE 3 
The effect of lime and acid phosphate on soil II 



TREATMENT 



No fertilizer 

Calcium oxide. . . . 
Ground limestone. 
Acid phosphate.. . 



AMOUNT 
PER ACRE 


CORN 9 


YEARS 


OATS 9 


YEARS 


Grain 


Stover 


Grain 


Straw 


pounds 


pounds 


pounds 


Pounds 


pounds 




51.50 


2759 


44.94 


1961 


1000 


57.33 


3149 


47.53 


2079 


1780 


54.84 


2820 


45.35 


1876 


320 


60.18 


3056 


46.16 


1912 



CLOVER 
8 YEARS 

Hay 

pou nds 

4074 
4580 
4362 
4277 



evaporation, was restored. This was continued for 12 weeks in order that 
the soil microorganisms should have time to adjust themselves to the changes 
in soil reaction. At the end of that time, the determinations of nitrifying 
power, ammonifying power, etc., were made. These determinations required 
about one-half of the soil. 



442 



FIRMAN E. BEAR 



Since the analyses showed that these soils were very deficient in total 
phosphorus, a thing which is commonly true of acid soils, it seemed advisable 
to apply phosphorus in a readily available form in order to remove it from being 
a possible limiting factor in the various bacterial activities studied. Accord- 
ingly, 0.2 per cent of mono-calcium phosphate, equivalent to 1000 pounds of 
phosphorus per 2,000,000 pounds of soil, was added, the moisture content was 
again restored, and the mixing was continued for another period of 12 weeks. 
At the end of this time, the above determinations were repeated. In some of 
the later experiments the calcium carbonate was added just previous to the 
time of studying the rate of nitrification, ammonification, etc. 



THE EFFECT OF CALCIUM CARBONATE ON THE NUMBER OF BACTERIA 

Soils I and II were used in these experiments, after they had received the 
various applications of calcium carbonate and had been mixed thoroughly 
each week for 12 weeks, as previously outlined. Plate counts of the number 
of microorganisms were made at the end of the 12-week period. After the 
0.2 per cent of mono-calcium phosphate had been added and mixed with the 
remainder of the soil each week for a second 12 weeks, plate counts were again 
made. Aliquots of the soil suspension were plated on Heyden agar. The 
plates were incubated at room temperature and counts were made at the end 
of 6 days. Table 4 shows the results of these counts. Each figure represents 
the average of four plates. 

As might be expected, the greatest relative change in the number of bac- 
teria occurred after the neutral point had been passed. This was true in 

TABLE 4 
The effect of calcium carbonate on the number of bacteria 



CALCIUM CARBONATE 




BACTERIA PER 


GRAM OF SOIL 




POUNDS OF son. 


Soil I without 
phosphorus 


Soil I with 
phosphorus 


Soil II without 
phosphorus 


Soil II with 
phosphorus 


pounds 













3,341,000 


4,150,000 


3,503,000 


3,438,000 


250 


4,127,000 


4,320,000 


3,418,000 


3,536,000 


500 


3,537,000 


3,540,000 


4,614,000 


4,421,000 


1,000 


3,439,000 


2,750,000 


3,781,000 


5,207,000 


2,000 


3,930,000 


2,520,000 


4,472,000 


5,781,000 


3,000 


4,127,000 


4,090,000 


4,919,000 


5,683,000 



Neutral point (Veitch method) 



4,000 


4,422,000 


5,820,000 


7,348,000 


10,005,000 


5,000 


5,306,000 


7,700,000 


9,741,000 


17,392,000 


7,500 


5,601,000 


7,070,000 


15,827,000 


14,297,000 


10,000 


3,341,000 


6,680,000 


14,973,000 


7,959,000 


20,000 


6,682,000 


10,750,000 


14,892,000 


3,635,000 


40,000 


9,335,000 


13,050,000 


18,199,000 


9,826,000 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 443 

both soils, as shown in figure 1. The 4000 and 5000-pound applications of 
calcium carbonate resulted in relatively large increases in numbers. Additions 
of calcium carbonate in excess of 7500 pounds per 2,000,000 pounds of soil 
gave somewhat uncertain results. There was a decrease in every case ac- 
companying an application of 10,000 pounds as compared with 7500 pounds 
of calcium carbonate. The 20,000 and 40,000-pound applications brought 
about marked increases in numbers. These fluctuations were probably due 
to the adjustment of the soil reaction to the point where it was more suitable 
to the requirements of some forms which developed in vast numbers under this 
optimum soil reaction. It seems quite evident that the application of calcium 
carbonate caused decided changes in the number of bacteria in these soils. 
The maximum increases in numbers had apparently not been reached in three 
of the four cases by applications of 40,000 pounds of calcium carbonate per 
2,000,000 pounds of soil. Similar trials with calcium oxide produced marked 
decreases in the number of bacteria in these soils following the larger appli- 
cations. This was probably due to the partial sterilizing action of calcium 
oxide previously referred to. 

THE EFFECT OF CALCIUM CARBONATE ON THE RATE OF AMMONIFICATION 

Soils I and II, after the treatments with calcium carbonate and mono- 
calcium phosphate previously referred to, were used in the experiments on 
ammonification. The source of nitrogen was Hammarsten's casein. Enough 
casein was added to supply 160 mgm. of nitrogen per 100 gm. of soil. The 
soil was then given an optimum moisture content and incubated in tumblers 
for 3 days at room temperature, after which analyses were made for ammonia 
by distillation with magnesium oxide. Each figure given in table 5 represents 
the average of two determinations which checked usually within less than 
1 mgm. per 100 gm. of soil. Other determinations, not reported in this paper, 
were made in which only 40 mgm. of nitrogen were added, with very satis- 
factory results. The author believes that 160 mgm. of nitrogen per 100 gm. 
of soil are likely to produce abnormal conditions in a soil, although in most of 
the ammonification experiments reported in the literature even larger amounts 
of nitrogen were supplied. 

The greatest relative increase in the rate of ammonification of casein, per 
unit of calcium carbonate applied, occurred with applications of 2000 pounds 
of calcium carbonate per 2,000,000 pounds of soil, as shown in figure 2. There 
was no marked increase in ammonification as the neutral point was passed. 
As. previously shown, this was also the case in the number of bacteria. Appli- 
cations of 250 pounds of calcium carbonate per 2,000,000 pounds of soil had a 
tendency to cause a decrease in the rate of ammonification. Applications of 
calcium carbonate in excess of 5000 pounds caused only a slight increase 
in the amount of ammonia produced. The 20,000 and 40,000-pound 
applications caused slight decreases in ammonia in several cases. There was 



444 



FIRMAN E. BEAR 



apparently no definite correlation between the number of bacteria and the 
amount of ammonia produced, although in general, increased amounts of 
calcium carbonate resulted in larger numbers of bacteria and more rapid 
ammonification. 



THE EFFECT OF CALCIUM CARBONATE ON THE RATE OF NITRIFICATION 

The effect of calcium carbonate on the rate of nitrification in soils I and II 
is shown in table 6. All figures in this and in succeeding tables of nitrification 

TABLE 5 

The effect of calcium carbonate on the rate of ammonification of casein* 



CALCIUM 


NITROGEN AS AMMONIA PER 100 GM. OF SOIL 


2,000,000 

POUNDS OF SOIL 


Soil I 

without 

phosphorus 


Soil I with 
phosphorus 


Soil Ilf 

without 

phosphorus 


Soil II 

without 

phosphorus 


Soil II with 
phosphorus 


Soil II with 
phosphorus 


pounds 



250 

500 

1,000 

2,000 

3,000 


mgm. 

72.40 
71.00 
72.80 
75.40 
78.50 
79.00 


mgm. 

60.70 
61.00 
59.60 
62.40 
68.00 
70.00 


mgm. 
38.85 
40.25 
45.36 
48.02 
57.54 
56.00 


mgm. 
22.89 
22.05 
29.05 
29.40 
41.65 
44.59 


mgm. 

29.51 

25.55 
28.00 
28.63 
43.40 
45.36 


mgm. 
39.20 
37.38 
37.80 
44.31 
55.44 
57.75 



Neutral point (Veitch method) 



4,000 


78.40 


70.60 


60.41 


45.85 


56.84 


63.00 


5,000 


76.30 


70.80 


67.27 


57.12 


64.61 


71.54 


7,500 


85.30 


74.80 


71.40 


57.05 


52.29 


67.97 


10,000 


83.60 


74.80 


74.48 


62.79 


63.07 


71.61 


20,000 


85.40 


77.80 


77.40 


60.76 


55.27 


68.81 


40,000 


87.50 


77.60 


77.42 


61.25 


51.61 


59.36 



* The results in each vertical column were obtained on the same day. Fluctuations in 
the temperature in the room are responsible for some of the differences observed in horizontal 
columns. 

f Four-day periods of incubation. 

represent averages of two determinations. As a rule, the duplicates agreed 
within less than 0.1 mgm. per 100 gm. of soil. Accordingly, only averages 
are reported. 

These soils were treated with calcium carbonate in varying amounts and 
with mono-calcium phosphate as previously outlined. At the end of the 12- 
week periods, samples of these soils of 100 gm. each were placed in 1000-cc. 
Erlenmeyer flasks for the nitrification experiments. To each flask were 
added 20 mgm. of nitrogen in the form of either ammonium sulfate or ammo- 
nium carbonate. After adding water to the optimum content, the soils were 
incubated for 21 days at room temperature, after which the nitrate determina- 
tions were made by the phenol-disulphonic acid method. 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



445 



A study of table 6 and figure 3 shows that the addition of calcium carbonate 
is followed by an increased nitrification which correlates almost directly with 
the increased application of calcium carbonate. This correlation holds fairly 
well in every case with applications up to 5000 pounds per 2,000,000 pounds 
of soil. There is no sudden break in the correlation as the neutral point is 
passed. Applications of calcium carbonate in excess of 5000 pounds are 
followed by increased nitrification, although the curve of increase begins to 
incline more toward the horizontal. In half of the experiments the curve 
was still ascending with applications of 40,000 pounds of calcium carbonate. 

TABLE 6 

The effect of calcium carbonate on the rate of nitrification 









NITROGEN 


AS NITRATE 


PER 100 GM. OF SOIL 






CALCIUM CARBON- 
ATE PER 
2,000,000 POUNDS 
OF SOIL 


Source 


sf nitrogen, 


ammonium 


sulfate 


Source of 


nitrogen, ammonium carbonate. 


Soil I* 
without 
phos- 
phorus 


Soil I 
with 
phos- 
phorus 


Soil II 
without 
phos- 
phorus 


Soil II 
with 
phos- 
phorus 


Soil I 
without 

phos- 
phorus 


Soil I 
with 
phos- 
phorus 


Soil II 
without 
phos- 
phorus 


Soil II 
with 
phos- 
phorus 


pounds 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 





1.08 


4.06 


5.28 


6.07 


1.38 


7.22 


5.29 


7.50 


250 


1.38 


4.32 


4.39 


5.34 


1.93 


8.24 


5.00 


8.00 


500 


1.43 


4.60 


4.40 


6.38 


2.11 


8.42 


5.42 


8.40 


1,000 


1.82 


5.24 


6.15 


6.75 


2.21 


9.52 


6.60 


8.55 


2,000 


2.29 


6.38 


8.50 


8.73 


3.01 


12.42 


9.03 


11.55 


3,000 


2.96 


9.34 


10.48 


10.43 


3.55 


15.30 


10.04 


12.50 



Neutral point (Veitch method) 



4,000 


3.13 


11.92 


15.74 


12.50 


3.28 


17.50 


11.87 


15.12 


5,000 


3.44 


13.86 


15.96 


15.00 


4.11 


18.00 


15.77 


18.75 


7,500 


3.48 


16.37 


18.18 


15.38 


4.69 


19.00 


17.27 


16.35 


10,000 


4.44 


19.35 


20.98 


15.98 


4.30 


20.00 


20.30 


16.16 


20,000 


4.00 


20.45 


22.87 


16.00 


4.32 


20.96 


19.80 


16.00 


40,000 


4.20 


22.55 


19.88 


15.00 


5.18 


23.30 


20.57 


15.00 



* An error was made in calculating the optimum moisture content and the soil in this 
experiment was too dry. 



This is directly contrary to the work of Scales previously referred to, which 
indicated that 50 per cent of the amount of calcium carbonate necessary 
to supply the lime requirement of the soil is sufficient to attain the maxi- 
mum rate of nitrification. Additional amounts are reported to have acted 
injuriously. 

The marked increase in the rate of ammonification observed with the 
addition of 2000 pounds as compared to 1000 pounds of calcium carbonate per 
2,000,000 pounds of soil was not followed by a coresponding increase in the 
nitrification. No correlation was found between the increased number of 
bacteria in the soil and the rate of nitrification except that in general the 



446 



FIRMAN E. BEAR 



application of increased amounts of calcium carbonate caused an upward 
tendency in the number of bacteria, as well as in the rate of nitrification. 
Since the agar plate method is not designed to include the nitrifying bacteria, 
no data are available as to the actual number of nitrifying organisms which 
were present in the soils following the applications of varying amounts of 
calcium carbonate. 



EFFECT OF CALCIUM CARBONATE ON THE RATE OF NITROGEN FIXATION BY 
NON-SYMBIOTIC SOIL ORGANISMS 

Samples I and II were employed again in these experiments after they 
had been treated as previously described. Shallow dishes having a depth of 
about 3 inches and a capacity of 400 gm. of soil were used for this work. Soil 

TABLE 7 

The effect of calcium carbonate on nitrogen fixation by non-symbiotic soil organisms 



CALCIUM CARBONATE PER 
2,000,000 


NITROGEN FIXED PER 100 GM. OF 


SOIL 


POUNDS OF son. 


Soil I without phosphorus 


Soil II without phosphorus 


Soil II with phosphorus 


pounds 


mgm. 


mgm. 


mgm. 





0.7 


0.3 


2.0 


250 


0.3 


0.8 


3.6 


500 


0.5 


0.8 


3.6 


1,000 


1.0 


0.6 


4.6 


2,000 


0.7 


1.4 


3.8 


3,000 


0.5 


2.3 


11.3 



Neutral point (Veitch method) 



4,000 


0.1 


2.8 


12.2 


5,000 


1.8 


4.1 


12.7 


7,500 


2.0 


6.0 


15.8 


10,000 


1.5 


5.8 


10.1 


20,000 


2.1 


4.5 


12.6 


40,000 


1.4 


. 4.4 


9.9 



from the various pots to which the calcium carbonate and mono-calcium 
phosphate had been applied was placed in the dishes and mixed thoroughly 
with 2 per cent of mannit. Optimum moisture conditions were secured and 
maintained as nearly as possible by adding water twice daily to restore that 
lost by evaporation. The soils were incubated 21 days at room temperature. 
After thorough drying, the entire samples were pulverized to pass a 100-mesh 
sieve and the total nitrogen was determined in triplicate. The triplicates 
agreed usually within 0.05 mgm. of nitrogen on 10-gm. samples. 

From a study of table 7, it appears evident that both calcium carbonate 
and mono-calcium phosphate were essential to the highest fixation of nitrogen. 
The mono-calcium phosphate had such a marked effect in increasing the nitro- 
gen-fixing power of soil II, as shown in figure 4, that it would seem that phos- 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 447 

phorus was equally as important as lime for the nitrogen-fixing organisms in 
this soil. The largest relative increase in nitrogen fixation followed an applica- 
tion of 3000 pounds of calcium carbonate per 2,000,000 of soil when accom- 
panied by the use of mono-calcium phosphate. Heavier applications of 
calcium carbonate caused an increase in nitrogen fixation until as much as 
10,000 pounds per 2,000,000 pounds of soil had been applied. This amount 
and heavier applications caused a decrease in nitrogen fixation. Apparently 
phosphorus was a limiting factor in nitrogen fixation in soil I, although time 
did not permit an experimental test of this point. The good effects resulting 
from the use of acid phosphate on these soils under field conditions may be 
due in part to this increased nitrogen fixation accompanying its use. This 
again is indicated by the analyses of the West Virginia Station fertility plots 
from which the author and others have shown that the plot receiving acid 
phosphate and sulfate of potash has accumulated 1173 pounds of nitrogen 
per acre during the last 15 years which could not be accounted for except by 
nitrogen fixation from the air. The evidence shown in table 7 indicates that 
calcium carbonate is necessary in addition to the phosphorus for the most 
effective nitrogen fixation. 

Following the suggestion of Christensen and Larsen (4), soil from each of 
the pots to which varying amounts of calcium carbonate had been applied, 
was used to inoculate Ashby's solution in order to study the relation between 
the film development on the surface of the liquid and the lime requirement of 
the soil. The brownish film was very well developed in the flasks inoculated 
with soil which contained an amount of calcium carbonate in excess of the 
requirement of the soil and practically disappeared as the quantity of calcium 
carbonate applied was reduced below the amount necessary to satisfy the 
requirement of the soil. The development of brown pigment is apparently 
closely related to the amount of lime in the soil and may be used as an index 
of the need of lime by the soil. Other experiments which will be discussed 
later indicated, however, that nitrogen fixation in Ashby's solution may take 
place when the solution is inoculated with soils having calcium-carbonate 
requirements as high as 4600 pounds per 2,000,000 pounds of soil. Apparently, 
the lack of development of the brownish film is not accompanied by the loss 
of ability to fix nitrogen, since no soil was found which did not show nitrogen 
fixation when inoculated into Ashby's solution and allowed to stand for 
21 days at room temperature. 

THE EFFECT OF CALCIUM CARBONATE ON THE FIXATION OF NITROGEN BY B. 
RADICICOLA OF THE SOYBEAN (SOJA MAX PIPER) 

Soils I and II were again used in these experiments. One-gallon pots were 
filled with these soils and the calcium carbonate was added. Each pot re- 
ceived an application of 0.2 per cent of mono-calcium phosphate. The pots 
were planted to soybeans, the beans having been previously inoculated with 



448 FIRMAN E. BEAR 

B. radicicola. Six beans were planted in each pot and later thinned to three 
per pot. After the beans had reached the stage where pods were formed, 
they were harvested, and records were taken of their green and dry weight, 
the number of nodules, the dry weight of nodules and the milligrams of nitrogen 
in the roots, tops and nodules. One crop was harvested from each pot during 
the summer of 1915 and another crop during the summer of 1916. The records 
are shown in tables 8 and 9. 

The number of nodules had a tendency to increase slightly with small appli- 
cations of calcium carbonate. Applications of more than 3000 pounds of calcium 
carbonate per 2,000,000 of soil caused a decrease in the number of nodules. 
This decrease was proportional to the amount of calcium carbonate applied. 
The dry weight of nodules was also decreased with large applications of cal- 
cium carbonate. The rate of decrease in dry weight of nodules with increased 
amounts of calcium carbonate was more marked than the rate of decrease 
in the number of nodules. The amount of nitrogen in the nodules was almost 
directly correlated with the dry weight of nodules, and decreased 'with addi- 
tional quantities of calcium carbonate. It will be noticed that in both cases 
the dry weight and total nitrogen of both stems and roots had a tendency 
to increase with small applications of calcium carbonate, but that applications 
in excess of 2000 pounds per 2,000,000 of soil had a tendency to cause a de- 
crease in dry weight and total nitrogen of the stems and roots. 

The total nitrogen fixed by soils I and II during the two years in which the 
two crops of soybeans were grown was determined. Analyses of the soil 
were made before and after the beans were grown. The difference in the 
nitrogen content of the soil at these two periods plus the nitrogen removed 
in the nodules, stems and roots, after subtracting the nitrogen content of the 
seed and water used in watering the plants, represents the nitrogen secured 
from the air. 

The total nitrogen fixed in two years per 2,000,000 pounds of soil, as shown 
in the last columns of tables 8 and 9, indicate that soil II has had a more active 
nitrogen-fixing flora than soil I. In so far as the chemical composition is 
concerned, the two soils correspond fairly well, as will be found by referring 
to the analyses of these two soils previously shown. By referring again to 
table 7, showing the rate of nitrogen fixation by Azotobacter , it will be seen 
that soil II was much more active in this respect than soil I. It is possible 
that a greater part of the nitrogen accumulated in soil II during the growing 
of the legumes was fixed in the soil through the agency of the non-symbiotic 
organisms. The nitrogen fixation had a tendency to decrease with applica- 
tions of calcium carbonate in excess of 2000 pounds per 2,000,000 pounds of 
soil, although the lime requirement of both soils indicated a need of 3500 
pounds. Apparently, with increased applications of calcium carbonate the 
rate of nitrification was so high, as indicated in table 6, that the soybeans were 
able to secure a greater part of their nitrogen in the form of nitrates. Large 
numbers of B. radicicola were present in all the pots whether treated with 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



449 



TABLE 8 
The effect of calcium carbonate on nitrogen fixation by B. radicicola of the soybean in soil I 





CALCIUM 

CARBONATE 

PER 

2,000,000 

POUNDS OF 

SOIL 


NUMBER 


NODULES 


STEMS 


ROOTS 


SOIL 


POT 


Dry 

weight 


Total 
nitro- 
gen 


Dry 

weight 


Total 
nitro- 
gen 


Dry 

weight 


Total 
nitro- 
gen 


Nitro- 
gen in 
begin- 
ning 
per pot 


Nitro- 
gen at 
end per 
pot 


Nitrogen 
fixed per 
2,000,000 
pounds of 
soil 


1 

2 
3 

5 
6 


pounds 



250 

500 

2,000 

3,000 


113 
67 
88 

100 

72 


mgm. 

653 
887 
749 
1017 
560 


mgm. 

31 
39 

33 
47 
27 


gravis 

13.2 
14.2 
14.3 
17.3 
14.9 


mgm. 

348 
377 
368 
464 
303 


grams 

4.3 

5.1 
4.4 
3.7 
3.4 


mgm. 

40 
42 
39 

35 
38 


grams 

3.0009 
3.0039 
3.0039 
3.0039 
3.0039 


grams 

2.8985 
2,8675 
2.8985 
2.8272 
2.7900 


pounds 

93 

98 

107 

130 

+8 











Neutral point (Veitch method) 








7 


4,000 


65 


317 


16 


11.9 


342 


2.9 


37 


3.0039 


2.7683 


-6 


8 


5,000 


79 


537 


26 


14.1 


363 


3.6 


39 


3.0039 


2.8923 


94 


9 


7,500 


84 


464 


22 


13.9 


426 


3.8 


46 


3.0039 


2.7218 


23 


10 


10,000 


66 


212 


11 


11.2 


342 


3.6 


52 


3.0039 


2.6660 


-60 


11 


20,000 


45 


220 


13 


10.3 


298 


5.1 


58 


3.0039 


2.6815 


-87 


12 


40,000 


57 


221 


13 


12.2 


383 


3.4 


50 


3.0039 


2.6505 


-56 



0.1727 gm. of nitrogen in the soybeans planted. 

0.0043 gm. of nitrogen in the water used in watering the soybeans. 

TABLE 9 

The effect of calcium carbonate on nitrogen fixation by B. radicicola of the soybean in soil II 





CALCIUM 

CARBONATE 

PER 

2,000,000 

POUNDS OF 

SOIL 


NUMBER 


NODULES 


STEMS 


ROOTS 


SOIL 


POT 


Dry 

weight 


Total 
nitro- 
gen 


Dry 

weight 


Total 
nitro- 
gen 


Dry 

weight 


Total 
nitro- 
gen 


Nitro- 
gen in 
begin- 
ning 
per pot 


Nitro- 
gen at 
end per 
pot 


Nitrogen 
fixed per 
2,000,000 
pounds of 
soil 


1 

3 
6 


pounds 



500 

3,000 


79 

75 
148 


mgm. 

781 
744 

577 


mgm. 

38 
37 
29 


grams 
14.8 
15.2 

13.5 


mgm. 

394 
440 

371 


grams 
4.1 
4.0 

4.7 


mgm. 

46 

42 
45 


grams 

2.6181 
2.6181 
2.6181 


grams 

3.2008 
3.1860 
3.1418 


Pounds 

570 

587 
511 











Neutral point (Veitch method) 








8 


5,000 


71 


330 


18 


12.3 


338 


3.2 


41 


2.6181 


3.1358 


476 


9 


7,500 


85 


251 


13 


12.7 


370 


3.8 


45 


2.6181 


3.1270 


490 


10 


10,000 


66 


207 


12 


11.8 


335 


3.3 


57 


2.6181 


3.0238 


408 


11 


20,000 


54 


124 


5 


11.5 


341 


4.0 


68 


2.6181 


2.9648 


377 


12 


40,000 


33 


126 


7 


14.4 


393 


3.0 


40 


2.6181 


2.9205 


265 



0.1727 gm. of nitrogen in the soybeans planted. 

0.0043 gm. of nitrogen in the water used in watering the soybeans. 



SOIL SCIENCE, VOL. IV, NO. 6 



450 



FIRMAN E. BEAR 



calcium carbonate or not, although quantitative determinations were not 
made of their numbers. 

THE EFFECT OF CALCIUM CARBONATE ON SOYBEANS UNDER FIELD CONDITIONS 

In order to determine whether soybean yields are increased by the use of 
calcium carbonate on soil I under field conditions, it was decided to grow 
soybeans on the fertility plots of the station farm during the summer of 1916. 
Three varieties of soybeans were sown in rows across the plots and cultivated 
during the growing season. One-half of each plot received an application of 
calcium carbonate at the rate of 2 tons per acre in the form of ground lime- 
stone. The yields of hay produced are given in table 10. The previous crop 
records and the analyses of the soils of these plots are given in tables 1 and li. 



TABLE 10 
Effect of ground limestone on yield of soybean hay on soil I 



19 

20 
21 
22 
26 
26 
28 
29 
31 
32 
34 
35 



TREATMENT 



N, P, K, CaO. 

M, CaO 

Check 

CaO 

M 

N,P,K 

P,K 

N, K 

N, P 

K 

P 

N 



CALCIUM- 

CARBONA1E 

REQUIREMENT 

PER 2,000,000 

POUNDS 



Pounds 





2800 



2800 

3200 

3600 

3400 

3200 

3600 

3400 

3400 



Averages . 



YIELD OF HAY PER ACRE 



No limestone 



pounds 

5270 
6390 
1605 
1920 
7150 
5300 
2820 
1285 
3375 
1495 
3285 
1220 



3426 



Limestone 



pounds 
5400 
6850 
1400 
2430 
7360 
5370 
4690 
2280 
4460 
1995 
4105 
1705 



4004 



INCREASE WITH 
LIMESTONE 



per cent 

+2 

+7 

-13 

+26 

+3 

+ 1 

+66 

+77 
+32 
+34 
+25 
+40 



+ 17 



From a study of the plots and the crop records, it would seem that the use 
of 2 tons of limestone per acre did not give sufficient increase in yield to justify 
the conclusion that soybeans will not grow well except on soils which have 
had their lime requirement satisfied. On plots 25 and 26, the soils of both of 
which have rather high calcium-carbonate requirements, but which also contain 
a fairly high content of nitrogen, the yield of soybeans was little affected by 
the limestone. This might mean that more nitrogen was secured from the 
soil on these plots and for this reason the crop was larger. However, the 
nodules were plentiful on the roots of the soybeans on plots 25 and 26 and, 
therefore, we could assume that nitrogen fixation from the air was taking place. 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



451 



EFFECT OF CALCIUM CARBONATE ON THE BACTERIAL ACTIVITIES OF DEKALB 
SOILS HAVING VARYING LIME REQUIREMENTS 

A large number of samples of acid soils all belonging to the Dekalb series 
were chosen from various parts of West Virginia and sent to the laboratory. 
From this number 12 samples were chosen which had calcium-carbonate 
requirements varying from 400 to 4600 pounds per 2,000,000 pounds of soil. 
The analyses of these soils are shown in table 11. These soils differ mostly 
because of the different systems of management practiced by the men who 
have farmed them since the areas from which the samples were chosen were 
cleared from the forest. Many of these areas had been farmed for from 
seventy-five to one-hundred years and others had not been farmed for more 
than a few years. 

TABLE 11 
A nalyses of soils of table 12 





POUNDS PER 2,000,000 POUNDS OF SOIL 




Nitiogen 


Phosphorus 


Carbon 


Calcium-carbonate 
requirement 




pounds 


pounds 


pounds 


pounds 


III 


3870 


1203 


41,420 


400 


IV 


1904 


586 


21,790 


1000 


V 


1669 


680 


17,600 


1200 


VI 


3374 


697 


47,230 


1400 


VII 


3142 


902 


32,140 


1600 


VIII 


2042 


1216 


20,280 


2000 


IX 


2602 


662 


32,450 


2200 


X 


4142 


1135 


48,680 


2600 


XI 


3384 


660 


39,490 


2800 


XII 


2750 


706 


32,140 


3200 


XIII 


1960 


608 


21,900 


3800 


XIV 


3124 


753 


48,280 


4600 



The rates of nitrification, ammonification, and nitrogen fixation were studied 
in an attempt to determine whether there was any relation between the activi- 
ties of the soil organisms and the calcium-carbonate requirements of these 
soils. 

In nitrification studies 100 gm. of soil to which varying amounts of calcium 
carbonate had been added were placed in 1000-cc. Erlenmeyer flasks and 
incubated with optimum moisture content at room temperature for 21 days, 
using ammonium sulfate as the source of nitrogen, adding a sufficient amount 
to supply 20 mgm. of nitrogen per 100 gm. of soil. In ammonification studies 
100-gm. samples of soil were used and varying amounts of calcium carbonate 
were added as in the nitrification tests. Casein was used as the source of 
nitrogen, 160 mgm. of nitrogen being added to 100 gm. of soil. The soil was 
incubated in tumblers at optimum moisture content for 3 days and the am- 



452 FIRMAN E. BEAR 

monia determined by distillation with magnesium oxide. Nitrogen-fixation 
tests were carried on by placing 10 gm. of soil in 100 cc. of Ashby's solution in 
800-cc. Kjeldahl flasks for a period of 21 days at room temperature. To 
one set of flasks enough calcium-carbonate was added to be equivalent to 
10,000 pounds per 2,000,000 pounds of soil. At the end of 21 days the total 
nitrogen was determined. All of the determinations on nitrification, ammoni- 
fication, and nitrogen fixation were performed in duplicate and these dupli- 
cates as a rule checked very closely. The results of these experiments are 
tabulated in table 12. 

In general the highest rates of ammonification occurred with soils having 
the lowest calcium-carbonate requirements. The applications of 2000 pounds 
and 5000 pounds of calcium-carbonate brought about marked increased in 
the rate of ammonification. Applications of 10,000 pounds of calcium car- 
bonate per 2,000,000 pounds of soil caused a decreased ammonification except 
in soils XIII and XIV, which had calcium-carbonate requirements of 3800 
and 4600 pounds, respectively. Apparently the application of 10,000 pounds 
of calcium carbonate per 2,000,000 of soil on soils having calcium-carbonate 
requirements of less than 3800 pounds is injurious to ammonifying organisms. 

There was no very definite correlation between the rate of nitrification of 
ammonium sulfate and the calcium-carbonate requirement of the soils. In 
general, the soils having high calcium-carbonate requirements had a very low 
nitrifying power. With soils having calcium-carbonate requirements in 
excess of 2200 pounds per 2,000,000 pounds of soil, the nitrifying organisms 
did not become markedly active even with large applications of calcium car- 
bonate. Either the nitrifying organisms were almost entirely absent or had 
become very inactive because of the unfavorableness of the medium in which 
they were living. 

There was no very marked correlation between the calcium-carbonate 
requirement of these soils and the nitrogen-fixing power of the soil organisms 
in Ashby's solution. Soil XIV, having a calcium-carbonate requirement of 
4600 pounds, was able to fix nitrogen to the extent of 2.9 mgm. per 100 cc. of 
Ashby's solution in 21 days. The rate of nitrogen fixation was increased in 
every case by the addition of calcium carbonate, but the effect was more 
marked on soils having a high requirement than in soils having a low calcium- 
carbonate requirement. It seems remarkable, however, that nitrogen fixa- 
tion took place in all cases even though some of the soils had very high lime 
requirements. 

EFFECT OF FERTILIZERS ON THE BACTERIAL ACTIVITIES OF SOILS 

These experiments were conducted in order to determine what effect differ- 
ences in the fertilizer treatments of the same soil would have on the bacterial 
activities in the soil. Samples of soil were chosen from 12 plots of the fertilizer 
series of the fertility plots on the West Virginia station, some of which differ 
considerably because of the fertilizer applications they have received during 
the last 15 years. Records of the treatments of the soil on these [plots have 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



453 



TABLE 12 

The effect of calcium carbonate on the activities of soil bacteria in Dekalb soils having varying 

lime requirements 



III 



CALCTUM- 

CARBONATE 

REQUIREMENT 

PER 2,000,000 

POUNDS OF SOU. 



pounds 



400 



IV 



VI 



vn 



VIII 



IX 



XI 



1,000 



1,200 



1,400 < 



1,600 



1,800 



2,200 { 



2,600 



2,800 { 



CALCIUM 

CARCONATE APPLIED 

PER 2,000,000 

POUNDS OF SOIL 



Pounds 



2,000 

5,000 

10,000 





2,000 

5,000 

10,000 



2,000 

5,000 

10,000 



2,000 

5,000 

10,000 



2,000 

5,000 

10,000 



2,000 

5,000 

10,000 

* 

2,000 

5,000 

10,000 



2,000 

5,000 

10,000 



2,000 

5,000 

10,000 



NITROGEN PER 100 GM. OF SOIL 



Nitrogen as 

ammonia from 

casein 



mgm. 

68.9 
76.5 
87.6 

82.4 



67.1 
81.2 
86.4 
74.6 

68.9 
80.5 
78.1 
72.6 

73.5 
83.2 
86.9 

87.7 

56.8 

77.3 
91.8 
87.5 

67.0 
76.2 
96.6 
87.3 

69.6 
83.1 
90.1 
82.7 

62.9 
75.1 
92.2 
91.8 

51.6 
68.3 
88.9 
92.2 



relative 

100 

111 

127 
118 



100 
121 
129 
111 

100 

117 
114 
105 



Nitrogen as 
nitrates from am- 
monium sulfate 



mgm. 

8.0 
12.6 
12.5 

9.4 



4.4 

8.4 

13.5 

14.0 

1.3 

7.8 

12.0 

16.0 



100 


5.8 


113 


7.3 


118 


8.0 


119 


12.6 


100 


1.5 


136 


3.0 


161 


5.7 


154 


16.0 


100 


0.4 


114 


1.5 


129 


8.0 


130 


15.7 


100 


0.8 


119 


5.1 


129 


9.8 


119 


4.6 


100 


2.7 


119 


4.8 


147 


5.2 


146 


13.0 


100 


0.8 


132 


2.2 


172 


1.7 


159 


2.1 



relative 

100 
158 
156 
118 



100 
191 
307 
318 

100 

600 

969 

1231 

100 
126 

138 
217 

100 

200 

380 

1067 

100 

375 
2000 
3925 

100 

638 

1225 

575 

100 
144 
156 
390 

100 

275 
213 
263 



NITROGEN FDCED IN 

100 CC. OF 
ASHBY'S SOLUTION 



mgm. 

7.6 



4.2 

5.6 

2.7 

4.1 
4.4 

5.6 
4.9 

5.9 
5.1 

6.5 
3.1 

4.7 
3.4 

4.8 
2.3 

3.2 



relative 

100 

114 

100 

133 
100 

152 
100 

127 
100 

120 
100 

127 
100 

151 
100 

141 
100 

139 



454 




FIRMAN E. 


BEAR 












TABLE 12— (Continued) 










CALCIUM- 
CARBONATE RE- 
QUIREMENT 
PER 2,000,000 
POUNDS OF SOIL 


CALCIUM 

CARBONATE APPLIED 

PER 2,000,000 

POUNDS OF SOIL 


NITROGEN PER 100 GM. OF SOIL 


KITROGE> 

100 c 

ashby's 


FIXED IN 


SOIL 


Nitrogen as 

ammonia from 

casein 


Nitrogen as 
nitrates from am- 
monium sulfate 


C. OF 
SOLUTION 




pounds 


pounds 


mgm. 


relative 


mgm. 


relative 


mgm. 


relative 




[ 





49.6 


100 


0.4 


100 


4.4 


100 


XII 


3,200 


2,000 
5,000 


70.5 
93.0 


142 

187 


1.4 
2.0 


350 
500 








. [ 


10,000 


89.5 


180 


3.2 


800 


5.6 


127 




f 





46.0 


100 


1.1 


100 


5.4 


100 


XIII 


3,800 | 


2,000 
5,000 


62.1 
76.3 


135 
166 


3.0 

4.5 


273 
410 








I 


10,000 


81.3 


177 


3.8 


345 


7.1 


131 




r 





30.0 


100 


0.3 


100 


2.9 


100 


XIV 


4,600 - 


2,000 
5,000 


45.5 
72.4 


152 
241 


0.7 
1.1 


233 
367 








- 


10,000 


86.3 


288 


0.7 


233 


4.4 


152 



been given in table 1 previously referred to. Analyses of the soil on the vari- 
ous plots were made and are recorded in table 13. 

The studies in nitrification, ammonification, and nitrogen fixation were 
conducted in the same manner as previously mentioned in the discussion of 
the 12 soils of the Dekalb series with varying calcium-carbonate requirements. 
It will be remembered that the soil of the fertility plots is also Dekalb soil. 
The records of these experiments are shown in table 14. 

TABLE 13 
.4 nalyses of soils of table 14 





TREATMENT 


POUNDS PER 2,000,000 POUNDS OF SOIL 


PLOT 


Nitrogen 


Phosphorus 


Carbon 


Calcium- 
carbonate 
requirement 






pounds 


pounds 


pounds 


pounds 


19 


N, P, K, CaO 


2130 


765 


' 24,500 





20 


M, CaO 


2700 


1045 


32,500 





21 


Check 


1830 


590 


21,200 


2800 


22 


CaO 


1750 


510 


19,400 





25 


M 


3240 


1220 


36,800 


2800 


26 


N, P, K 


2665 


900 


30,400 


3200 


28 


P,K 


2280 


850 


26,000 


3600 


29 


N, K 


2290 


640 


27,000 


3400 


31 


N, P 


2395 


880 


28,000 


3200 


32 


K 


2310 


740 


29,200 


3600 


34 


P 


2300 


885 


28,200 


3400 


35 


N 


2100 


620 


28,800 


3400 



N indicates nitrate of soda; P, acid phosphate; K, sulfate of potash; M, manure. 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



455 



TABLE 14 
The effect of calcium carbonate on the activities of soil bacteria in Dekalb soils which have received 

varying fertilizer treatments 





CALCTUM- 
CARBONATI 
REQUIRE- 
MENT PER 
2,000,000 
POUNDS OF 

son. 


CALCIUM 

CARBONATE 

APPLIED 

PER 

2,000,000 

POUNDS OF 

SOIL 


NITROGEN PER 


100 GM. OF son. 


NITROGE] 

100 

ashby's 




TREATMENT 


Nitrogen as 

ammonia from 

casein 


Nitrogen as 
nitrates from am- 
monium sulfate 


* FIXED IN 
CC. OF 
SOLUTION 




pounds 


Pounds 


mgm. 


relative 


mgm. 


relative 


mgm. 


relative 









78.3 


100 


15.3 


100 


5.6 


100 


N, P, K, CaO 


I 


2,000 
5,000 


80.1 
78.3 


102 
100 


18.5 
19.5 


121 
127 










10,000 


77.4 


99 


20.3 


133 


7.5 


134 









87.8 


100 


17.5 


100 


6.2 


TOO 


N, CaO 


I 


2,000 
5,000 


89.5 
87.0 


102 
99 


21.5 
22.5 


123 
129 










10,000 


84.3 


96 


22.0 


126 


6.4 


103 









60.6 


100 


1.2 


100 


3.7 


100 


Check 


2,800 J 


2,000 
5,000 


65.8 
78.2 


108 
129 


5.4 
11.8 


450 

983 














10,000 


79.7 


131 


15.5 


1275 


4.2 


113 









71.8 


100 


8.5 


100 


2.6 


100 


CaO 


I 


2,000 
5,000 


75.2 
82.9 


105 
115 


15.8 
18.3 


187 
215 














10,000 


82.7 


115 


22.0 


259 


4.8 


185 









71.7 


100 


6.7 


100 


5.1 


100 


M 


2,800 


2,000 
5,000 


75.3 
90.5 


105 
126 


12.5 
16.3 


186 
243 














10,000 


90.0 


125 


21.5 


321 


7.2 


141 









70.1 


100 


2.9 


100 


4.2 


100 


N,P,K 


3,200 J 


2,000 
5,000 


80.3 

85.5 


114 
122 


7.0 
9.3 


241 
321 










10,000 


86.6 


126 


13.8 


475 


6.1 


145 









58.4 


100 


1.4 


100 


4.3 


100 


P, K 


3,600 1 


2,000 
5,000 


66.2 
76.3 


109 
131 


4.3 

8.5 


301 
601 














10,000 


82.4 


141 


13.0 


928 


5.5 


128 




f 





56.6 


100 


1.5 


100 


5.1 


100 


N, K 


3,400 J 


2,000 
5,000 


65.9 

75.5 


116 
133 


4.8 
7.0 


320 

467 














10,000 


81.8 


144 


10.0 


667 


6.8 


133 




( 





60.9 


100 


1.8 


100 


4.4 


100 


N, P 


3,200 J 


2,000 
5,000 


68.7 
82.9 


113 
136 


5.8 
9.7 


322 
504 








1 


10,000 


85.2 


140 


12.8 


701 


5.5 


125 



456 



FIRMAN E. BEAR 



TABLE 14— (Continued) 





CALCIUM- 
CARBONATE 
REQUIRE- 


CALCIUM 

CARBONATE 

APPLIED 


NITROGEN PER 100 GM. OF SOIL 


NITROGEN 












TREATMENT 


MENT PER 

2,000,000 

POUNDS OF 
SOIL 


PER 

2,000,000 

POUNDS OF 

SOIL 


Nitrogen as 

ammonium from 

casein 


Nitrogen as 
nitrates from am- 
monium sulfate 


100 CC. OF 
ASHBY'S SOLUTION 




pounds 


pounds 


mgm. 


relative 


mgm. 


relative 


mgm. 


relative 




' 





47.9 


100 


1.1 


100 


7.4 


100 


K 


3,600 < 


2,000 
5,000 


57.4 
75.7 


120 

158 


2.5 
5.0 


237 
454 














10,000 


84.0 


174 


6.6 


600 


7.6 


103 









49.5 


100 


1.1 


100 


5.3 


100 


P 


3,400 i 


2,000 


65.0 


131 


3.4 


301 










5,000 


79.4 


160 


7.0 


636 








. 


10,000 


84.3 


170 


8.5 


772 


7.6 


143 




c 





50.2 


100 


1.1 


100 


3.8 


100 


N 


3,400 I 


2,000 


64.9 


129 


2.9 


272 










5,000 


78.6 


156 


5.5 


500 








• 


10,000 


83.1 


165 


7.3 


663 


6.9 


182 



Nitrification of ammonium sulfate was not very active in these soils except 
on the plots where lime had been applied in the field. Even the soil of plots 
26, 28 and 31, which had been producing very satisfactory crops as indicated 
in table 1, did not contain vigorous nitrifying organisms. The rate of nitri- 
fication was materially increased by applications of calcium carbonate. The 
nitrifying organisms were much more active in the soil from the manure plots 
than in the soil from any of the other plots except where lime had been applied. 
There was a general tendency for the rate of ammonification of casein to 
decrease with an increase in the lime requirement of the soils. There were some 
marked exceptions to this tendency, notably plots 25 and 26. A study of the 
analyses of these plots shows a high total content of nitrogen and organic 
matter. No lime has ever been applied to plots 25 and 26. This increased 
nitrogen in the form of protein represents an increased amount of material 
available for the action of ammonifying organisms. If a large amount of 
nitrogen has been stored up in the soil, the amount of ammonia produced 
without any applications of calcium carbonate would be sufficient to produce 
satisfactory yields of those crops which are able to utilize ammonia, on a soil 
having lime requirements no higher than those of plots 25 and 26. The 
tendency for small applications of calcium carbonate to be relatively much 
more effective than larger applications was again shown in these experiments. 
It was evident that ammonification proceeds fairly satisfactorily without the 
application of calcium carbonate, especially, as suggested in the preceding 
discussion, if the content of organic nitrogen is high. Large applications of 
calcium carbonate had a tendency to reduce the rate of ammonification. 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



457 



Nitrogen fixation took place very readily in Ashby's solution when inoculated 
with soil from any of the plots. There did not seem to beany correlation be- 
tween the calcium-carbonate requirement and nitrogen fixation. The addi- 
tion of calcium carbonate to Ashby's solution caused an increase in nitrogen 
fixation in every case, but this increase was no more marked in soil having a 
high lime requirement than in soil having a low lime requirement. 




2000 



300O 4OO0 SOOO 7500 >0.O0O 

Pounds calcium carbonate per 2,000,000 pounds soil. 

Soiilf with phosphorus SoUl with phosphorus. 

Soil II without phosphorus Soil 1 without phosphorus. 

Fig. 1. The Effect of Calcium Carbonate on the Number of Bacteria in Soils I 

and II 



SUMMARY AND CONCLUSIONS 

This investigation was undertaken as a preliminary step in the study of the 
possibilities of a system of acid agriculture on soils somewhat distantly re- 
moved from a source of lime. A study was made of the relation between the 
activities of the soil bacteria concerned in nitrogen accumulation and nitrogen 
transformations and the lime requirement of certain soils. The lime require- 
ment of these soils varied from none to 4600 pounds of calcium carbonate per 
2,000,000 pounds of soil. To different portions of these soils calcium carbon- 
ate was added in amounts ranging from 0.01 per cent to 2 per cent of the weight 



458 



FIRMAN E. BEAR 



of the soil. The data accumulated show that the various groups of soil 
organisms vary in their response to applications of calcium carbonate. 

Ammonification proceeded fairly satisfactorily in most of the soils without 
the application of lime. The use of moderate amounts of calcium carbonate 
increased the rate of ammonification in most cases. Small applications were 
much more effective, relatively, than large applications. 









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IOOO 2000 300O 4000 5000 7500 IQOOO 

Pounds calcium carbonate per 2,000,000 pounds soil. 
Soil I - Soil II 

— Soil II w'lth four day period of incubation. 

Fig. 2. The Effect of Calcium Carbonate on the Rate of Ammonification in Soils I 

and II with Phosphorus 



The rate of nitrification was almost directly correlated with the amount of 
calcium carbonate supplied. Excessive applications were not injurious to the 
nitrifying organisms. Soils having high lime requirements showed practically 
no nitrification until calcium carbonate had been mixed with them. 

Nitrogen fixation by non-symbiotic soil organisms was considerably in- 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 



459 



creased by the addition of calcium carbonate. The application of mono- 
calcium phosphate also was necessary for maximum nitrogen fixation. All 
of the soils studied accumulated considerable amounts of nitrogen when in- 
cubated in Ashby's solution without the addition of calcium carbonate, 
although its use increased the rate of nitrogen fixation. 

A lime requirement of 3000 pounds was not sufficient to prevent a good 
growth of soybeans on soil well fertilized with acid phosphate or manure. 
Nitrogen fixation accompanying the growth of soybeans took place readily 




s 



/ 



Soil I with ammonium carbonate. 

Soil U with ammonium carbonate. 
Soil II with ammonium sulphate. 
Soil I with ammonium sulphate. 



2000 3000 4000 SOOO 7 ^ 00 

Pounds calcium carbonate per 2, 000, coo pounds soil 



10000 



Fig. 3. The Effect of Calcium Carbonate on the Rate of Nitrification in Soils I 

and II with Phosphorus 



in acid soils. This fixation was increased by small applications but decreased 
by large applications of calcium carbonate. 

From these facts the following conclusions seem justified: 

1. Plants which are able to utilize ammonia nitrogen need not suffer from 
nitrogen hunger when grown on soils having lime requirements no higher 
than those studied in these investigations. 

2. Plants which depend on nitrates as their source of nitrogen may suffer 
from the lack of available nitrogen in soils having high Ume requirements, 
unless these requirements have been at least partially satisfied. 



460 



FIRMAN E. BEAR 



3. The supply of nitrogen in acid soils may be maintained by growing acid- 
resistant legumes, of which the soybean is one. Undoubtedly, the use of 
acid phosphate aids materially in the nitrogen-fixation processes in acid soils. 

4. Small applications of calcium carbonate are, as a rule, relatively more 
effective than large applications as a means of increasing the bacterial activities 
in acid soils. 

Acknowledgment is due Dr. E. B. Fred of the University of Wisconsin 
for many helpful suggestions and criticisms offered during the progress of 
this investigation. 



16 r 



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Pounds calcium carbonate per 2,000,000 pounds of soil. 
— With phosphorus Without phosphorus. 



Fig. 4. The Effect of Calcium Carbonate on Non-Symbiotic Nitrogen Fixation in 

Soil II 

REFERENCES 



(1) Ashby, S. F. 1907 Some observations on the assimilation of atmospheric nitrogen 

by a free-living soil organism, Azotobacter Chroococcum of Beijerinck. In 
Jour. Agr. Sci., v. 2, p. 35. 

(2) Bear, F. E., and Salter, R. M. 1916 The residual effects of fertilizers. W. Va. 

Agr. Exp. Sta. Bui. 160, 25 p. 

(3) Bear, F. E., and Salter, R. M. 1916 Methods in soil analysis. W. Va. Agr. Exp. 

Sta. Bui. 159, 24 p., 2 fig. 



BACTERIAL ACTIVITY AND LIME REQUIREMENT 461 

(4) Christensen, H. R., and Laksen, O. H. 1911 Untersuchung ttber Methoden zur 

Bestimmung des Kalkbedurfnisses des Bodens. In Centbl Bakt. [etc.], Abt. 2, 

Bd. 29, p. 347. ■ . . 

(5) Clark W M., and LtJbs, H. A. 1917 The colorimetnc determination of hydrogen- 

i'on concentration and its applications in bacteriology. In Jour. Bact., v. 2, 
p. 1-11, 109-136, 191-236. _ 

(6) Coville, F. V. 1913 The agricultural utilization of acid lands by means of acid 

tolerant crops. U. S. Dept. Agr. Bui. 6, 13 p. 

(7) Drew G H 1914 On the precipitation of calcium carbonate in the sea by marine 

~ bacteria and on the action of denitrifying bacteria in tropical and temperate 
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(8) Fischer, H. 1909 Ueber den Einfluss des Kalkes auf die Baktenen ernes Bodens. 

/wLandw.Vers. Stat., Bd. 70, p. 335. 

(9) Frear W. 1915 Sour soils and liming. Penn. Dept. Agr. Bui. 261, 221 p. 

(10) Fred, E. B., and Graul, E.J. 1916 The gain in nitrogen from the growtn of legumes 

'on acid soils. Wis. Agr. Exp. Sta. Research Bui. 39, p. 37. 

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Soc. [London], v. 94, p. 524. 

(13) Hall, A. D., and Miller, N. H. J. 1911 The production of acids and alkalies in the 

'soil. In Jour. Chem. Soc. [London], v. 100, p. 35. 

(14) Harter, L. L. 1910 Investigations. Va. Truck Exp. Sta Bui. 4, p. 80 

15 )» Heinrich, R. 1892 Mergel und Mergeln auch die Chemische Bodenanalyzen und 
ihre Bedeutung fur die Feststellung der Dungbedurftigkeit der Bodens. In 

Centbl. Agr. Chem. 1892. 

(16) Hoffmann, C. 1906 Relation of soil bacteria to nitrogenous decomposition. Wis. 

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(17) Hoffmann, C, and Hammer, B. W. 1910 Some factors concerned in nitrogen fixa- 

tion by Azotobacter. Wis. Agr. Exp. Sta. Research Bui. 12, p. 172 

(18) Hopkins, C. G. 1910 Soil Fertility and Permanent Agriculture. Ginn and Co., 

New York, p. 214. n , , .. 

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(20) Hutchinson, H.B., and Miller, N.H. J. 1911 The direct ^^°^°^ 

and organic forms of nitrogen by higher plants. In Centbl. Bakt. (etc.), Abt. 2, 
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(21) Itano, A. 1916 The relation of hydrogen-ion concentration of media to he pro- 

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(22) Kellerman, K. F., and Robinson, T. R. 1910 Legume inoculation and litmus reac- 

tion of soils. U. S. Dept. Agr. Bur. Plant Indus. Cir. 71, 11 p. 

(23) Kellerman, K. F., and Smith, N. R. 1914 Bacterial precipitation of calcium car- 

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(24) Kelley, W. P. 1911 The assimilation of nitrogen by nee. Hawaii Agr. Exp. bta. 

(25) KoPEL E OFF, 2 N. P 'm 2 6 'The effect of soil reaction on ammonification by certain soil 

fungi. In Soil Sci. v. 1, p. 571 influen ce of calcium carbonate 

(26) Kossovitch, P. S., and Althausen, L. 1907 The influence oi ca 

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(27) Lvon/t'l.^a'nd'bizzel^J.A. 1913 Some relations of certain higher plants to the 

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1 Reference not verified. 



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(28) Miller, F. 1914 Ueber den Einfluss des Kalkes auf die Bodenbakterien. In Ztschr. 

Garungsphysiol., Bd. 4, p. 194-206. 

(29) Scales, F. M. 1915 Relation of lime to production of nitrates and mineral nitrogen. 

Abs. in Science, n. s., v. 42, p. 317. 

(30) Sharp, L. T., and Hoagland, D. R. 1916 Acidity and adsorption in'soils as measured 

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(31) Temple, J. G. 1914 Nitrification in acid or non-basic soils. Ga. Agr. Exp. Sta. 

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(32) Truog, E. 1916 (a) A new apparatus for the determination of soil carbonates and 

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(33) Truog, E. 1916 (b) The cause and nature of soil acidity with special regard to 

colloids and adsorption. In Jour. Phys. Chem., v. 20, p. 457-484. 

(34) Veitch, F. P. 1904 Comparison of methods of estimation of soil acidity. In Jour. 

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(36) Wheeler, H. J. 1897 The fifth year's observation upon the growth of plants upon 

an acid upland soil, limed and unlimed. R. I. Agr. Exp. Sta. Rpt., 1897, p. 
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(37) Wheeler, H. J. 1903 A further study of the influence of lime upon plant growth. 

R. I. Agr. Exp. Sta. Bui. 96, 44 p. 

(38) Wheeler, H. J. 1914 The comparative effect on different kinds of plants of liming 

on acid soil. R. I. Agr. Exp. Sta. Bui. 160, p. 407-446. 

(39) Whiting, A. L. 1913 Soybean and cowpea nodules, hi Breeder's Gaz., v. 64, p. 

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Sta. Bui. 230, p. 1. 

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published data). 



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